条石鲷的早期生长发育特征

对条石鲷(Oplegnathus fasciatus)早期生活史阶段生长发育特征进行了观察和测量,描述了胚胎和胚后各发育阶段的生长发育特征。条石鲷成熟卵子为浮性、端黄卵,卵径0.86mm±0.012mm(n=30),单油球,油球径0.18mm-0.22mm。在水温23.5℃±0.5℃、盐度30.5、pH8.0-8.3条件下,受精卵经27.5h孵化出膜。初孵仔鱼消化道细而直,随着仔鱼发育,消化道变得粗大、弯曲,肠道内褶回增多,消化能力增强。3日龄(仔鱼孵化出膜后第一天称为0日龄,以此类推)仔鱼卵黄囊消耗殆尽,开口,肛门与外界相通,油球在6日龄消耗完毕,进入外源性营养阶段。仔稚鱼培育在室内水泥池进行。在水温21℃-24℃、盐度27-33、pH8.0-8.3条件下,仔鱼开口饵料为S型褶皱臂尾轮虫,4日龄仔鱼开始摄食轮虫或轮虫卵,13日龄开始摄食卤虫无节幼体,25日龄开始进行配合饲料转化,30日龄后苗种摄食配合饲料良好。4日龄仔鱼鳔原基形成,7日龄时鳔开始充气变为亮泡状,生活史终生有鳔。色素细胞首先出现在12h50min的胚体上,至18h已在胚体全身分布,随着胚体发育,色素细胞增多。脊椎骨末端弯曲在15日龄开始,至24日龄弯曲过程完成,除腹鳍外其它鳍的鳍条数与成鱼一致,牙齿(硬齿和绒毛齿)生成,可咬食饵料。体表七条横向黑色素带是条石鲷的一个显著体态特征。21日龄鱼体表形成第一条横向黑色素带,23日龄时体表形成第二条色素带,25日龄时形成第三条色素带,29日龄时仔鱼体表第五条色素带形成,至40日龄时第七条色素带形成,此时稚鱼体态除背鳍、尾鳍、腹鳍上色素外与成体相似。60日龄幼鱼鳞片形成,在体态上与成体无明显区别。     

盐度对条石鲷胚胎发育的影响

为了确定条石鲷胚胎孵化的适宜盐度,在试验水温为23.0℃～25.2℃条件下,设置了8个盐度组进行条石鲷胚胎孵化试验,研究了盐度对条石鲷胚胎发育的影响。结果表明:低盐度和高盐度对胚胎有持续性伤害,均可造成胚胎和卵黄球在卵裂期收缩而死亡,一部分胚胎在原肠期之后收缩死亡;低盐度可造成初孵仔鱼畸形率增加,主要引起仔鱼脊柱\"L\"形和\"C\"形弯曲,而高盐度可导致胚体胚孔关闭以后尾部的畸形发育;通过分析各盐度组的孵化率、仔鱼畸形率和盐度之间的关系,得出健康仔鱼比例(PHL)和盐度(S)的关系式为PHL=-0.0018S2-0.1135S-0.8853(R2=0.948),以PHL70%为适宜孵化盐度范围,以PHL90%为最适孵化盐度范围,由此确定在23.0℃～25.2℃条件下,条石鲷的适宜孵化盐度范围为21～42(PHL70%),最适为30～33(PHL90%)。     

条石鲷仔、稚、幼鱼脂肪酸组成变化研究

通过采用GC/MS法研究了条石鲷(Oplegnathus fasciatus)仔鱼、稚鱼及幼鱼阶段的脂肪酸组成和变化特点。共检测到28种脂肪酸,其中饱和脂肪酸(SFA)13种,单不饱和脂肪酸(MUFA)7种,多不饱和脂肪酸(PUFA)8种。结果表明:条石鲷鱼苗内源性营养阶段以饱和脂肪酸C16∶0、C20∶0及单不饱和脂肪酸C16∶1、C18∶1作为能量代谢的主要来源;必需脂肪酸C20∶4(n-6)(AA)、C22∶5(n-3)(DPA)和C22∶6(n-3)(DHA)在稚鱼期含量较低,∑EPA+DHA仅为6.89%,认为是发生稚鱼胀鳔病的主要原因;仔鱼开口前体内的DHA和EPA是由母体卵黄提供的。     

条石鲷雌雄鱼核型及C-带的比较分析

染色体核型和带型分析是鱼类细胞遗传学研究的重要内容之一,不仅对认识鱼类的分类系统、进化关系及染色体演化过程具有重要意义,还可为鱼类遗传育种提供细胞遗传学依据。我国从20世纪70年代开始开展鱼类细胞遗传学方面的研究,到目前为止,有关鱼类染色体核型已有很多报道。在海水鱼类中,据不完全统计,至2006     

消化酶在条石鲷成鱼体内的分布及pH对消化酶活力的影响

采用酶学分析方法研究了消化酶在条石鲷(Oplegnathus fasciatus)成鱼体内不同消化器官中的分布和pH对其消化酶活力的影响。结果表明,(1)蛋白酶活力为胃>后肠>中肠>前肠>肝,淀粉酶活力为前肠>中肠>后肠>胃>肝,脂肪酶活力为前肠>中肠>胃>后肠>肝,表明胃是消化蛋白类物质的主要场所,肠道在各种营养物质的消化中起重要作用,而肝中3种酶活力很低,可能在食物的消化中作用较小。(2)条石鲷胃的蛋白酶和淀粉酶的最适宜pH值分别为3.2和5.6,胃蛋白酶在强酸性条件下活力较高,而胃淀粉酶在弱酸性条件下活力较高;肝的蛋白酶和淀粉酶的最适pH是7.6,在中性条件下活性较高;肠的蛋白酶和淀粉酶的最适pH为6.6,在弱酸性条件下活力较高。     

不同盐度下条石鲷胚胎及卵黄囊仔鱼的形态变化

为了研究盐度对条石鲷(Oplegnathus fascltus)胚胎及卵黄囊仔鱼生长发育的影响,将条石鲷的受精卵置于不同盐度的水体中, 观察盐度胁迫下条石鲷胚胎的沉浮情况、卵径、油球径、孵化率、畸形率, 以及孵化后卵黄囊仔鱼的形态变化和营养物质的消耗状况。结果表明:条石鲷受精卵在盐度25(含25)以下的水体中为沉性, 在盐度30(含30)以上的海水中全部漂浮于水表层, 在对照组盐度26的水体中大部分胚胎浮在水上层;在水温22.5 ℃～24.0 ℃的条件下, 条石鲷受精卵经24～30 h可孵化成仔鱼, 盐度对孵化时间影响不显著(P＞0.05);盐度为20～50都能孵出仔鱼, 但孵化率和畸形率因盐度不同而有显著差异;综合分析表明,条石鲷受精卵孵化的适合盐度范围为25～35,理论上的最适孵化盐度范围为28.5～30.5;受精卵卵径与孵化盐度呈反比,盐度与卵黄囊被吸收程度呈正比,与全长和体高呈反比,油球径不因盐度不同而改变。     

云斑尖塘鳢肿大细胞病毒属虹彩病毒的分离与鉴定

2009年10月, 广东顺德地区一云斑尖塘鳢养殖场暴发不明病因疾病, 发病尖塘鳢体长15-18 cm不等,死亡率约85%, 濒死尖塘鳢从池塘底层游至水面, 呈现游动失衡状态直至死亡。死亡尖塘鳢腹部膨大,剖检可见肝脏、脾脏、肾脏肿大, 有出血斑点, 从内脏器官肝脏、脾脏和肾脏未分离到致病菌。病鱼内脏组织研磨过滤除菌后,腹腔注射20尾尖塘鳢, 7d后开始出现死亡, 10d后全部死亡, 对照组无死亡。自然发病鱼和人工感染鱼的病理切片显示肝脏、脾脏和肾脏出现大量肿大细胞,超薄切片经电子显微镜观察, 肝脏、脾脏和肾脏观察到大量病毒颗粒。电镜下病毒呈六边形,直径约135 nm,形态与虹彩病毒相似。针对虹彩病毒主衣壳蛋白(Major capsid protein,MCP)序列设计引物,提取自然发病鱼和人工感染鱼的DNA作为模板, 均能扩增出预期大小的特异性产物。利用NCBI的Blast搜索, 结果显示扩增序列与肿大细胞病毒属的传染性脾肾坏死病毒(ISKNV)、闪电丽鱼虹彩病毒(DGIV)和条石鲷虹彩病毒(RBIV)MCP核苷酸序列同源性分别为98.8%、98.1%和94.7%。利用MCP序列构建的系统发育树显示, 导致云斑尖塘鳢发病死亡的病毒为肿大细胞病毒属虹彩病毒, 暂命名云斑尖塘鳢虹彩病毒(MSGIV)。     

水生动物虹彩病毒的分子生物学

近年来在世界范围出现的由虹彩病毒感染引起的水生动物高发病率和高死亡率现象给水产业造成了重大经济损失,引起了病毒学家们的热切关注,根据ICTV第七次报告,虹彩病毒科(Iridoviridae)分为4个属,包括感染脊椎动物的蛙病毒属(Ranavirus)和淋巴囊肿病毒属(Lymphocystivirus),以及感染无脊椎动物的虹彩病毒属(Iridovirus)和绿虹彩病毒属(Chloriridovirus).     

虹彩病毒囊膜蛋白的研究进展

虹彩病毒是一类含有线性双链DNA的二十面体病毒,其感染可导致水产养殖生物（如鱼类）等严重疾病。囊膜蛋白在病毒吸附、进入以及释放过程中起着关键作用,阐明囊膜蛋白的结构和功能有助于揭示虹彩病毒入侵的分子机制。本文通过对虹彩病毒囊膜蛋白的基本特征、表达、亚细胞定位、功能、相互作用以及疫苗开发等方面进行阐述,旨在为未来的研究工作提供新的思路。     

低等水生脊椎动物虹彩病毒的免疫逃逸策略

虹彩病毒是一类大分子双链DNA病毒,目前证实可感染100多种水生动物,已给水产养殖业造成重大经济损失,同时也危及到野生动物种群的生物多样性及生态平衡。虹彩病毒在长期的病原与宿主相互作用及进化过程中发展形成了相当系统和完善的免疫逃逸策略,以逃避免疫攻击,完成在宿主体内的复制,以及种内和种间传播。综合归纳分析近年来国内外有关低等脊椎动物虹彩病毒免疫逃逸策略,以及宿主对这些病原的先天性免疫反应研究进展。     

Isolation and Characterization of Male-Specific DNA Markers in the Rock Bream Oplegnathus fasciatus

Sex-specific DNA markers applicable were very useful for elucidating the sex-determination mechanism and sex control in fishes. In the present study, amplified fragment-length polymorphism (AFLP) approach with 144 primer combinations was employed to identify sex-specific markers in the rock bream. Four male-specific AFLP fragments were identified which were designated as Opl286, Opl237, Opl422, and Opl228. Further sequence analysis of the sex markers’ genomic region revealed subtle differences between the males and females. We identified four male-specific single-nucleotide polymorphisms (SNPs) and a deletion of 8 bp in marker Opl286, six male-specific SNPs in marker Opl237, three male-specific SNPs in marker Opl422, and eight male-specific SNPs and 1 bp inversions in marker Opl228. Specific primers were designed based on the nucleotide variation in the sequences to develop a simple polymerase chain reaction method for identifying the genetic sex of rock bream. As a result, three out of the four male-specific markers were converted into SNP markers. The male-specific AFLP markers and AFLP-derived SNP markers were tested in 100 individuals collected from three locations around the coast of Zhoushan, yielding reproducible sex identification. These male-specific DNA markers are a useful tool for the identification of the sex-determining locus in rock bream and for guiding artificial breeding programs.     

Protective effect of herbal and probiotics enriched diet on haematological and immunity status of Oplegnathus fasciatus (Temminck & Schlegel) against Edwardsiella tarda

This study determines the effect of diet enriched with the herb Baical skullcap Scutellaria baicalensis, and/or probiotics Lactobacillus sakei BK19 in rock bream, Oplegnathus fasciatus (32 ± 3 g) against Edwardsiella tarda. The changes in haematological parameters, innate immune response, and disease resistance were investigated after 1, 3, and 6 weeks. The white blood cell count (WBC: 10(4) mm(-3)), red blood cell count (RBC: 10(6) mm(-3)), and haemoglobin (Hb: g dl(-1)) levels significant increased (P < 0.05) with mixed diet on 3rd and 6th week and probiotics enriched diet on 6th week. The haematocrit (Ht: %) level significantly increased (P < 0.05) when fed with mixed diet on weeks 1-6. Interestingly, in mixed diet group the lymphocytes (LYM), monocytes (MON), and neutrophils (NEU) significantly increased from week 1-6. The eosinophils (EOS) significantly increased in all the treated groups. In the probiotics or mixed diet groups the total protein (TP: g dl(-1)) increased significantly on weeks 3 and 6. The serum lysozyme activity significantly was enhanced in all the treated groups indicating an increase in the innate immunity level. Serum complement, antiprotease activities, reactive oxygen species (ROS) and reactive nitrogen species (RNS) production significantly increased from week 1-6 with mixed diet. The maximum protection against E. tarda was recorded in mixed diet group with a minimum cumulative mortality of 20% and a high relative percent survival (RPS) of 72.84. In the probiotics and herbal diet groups the cumulative mortality was 25% and 35% and RPS was 68.63 and 59.42, respectively. This study indicates that administration of probiotics or mixed diets can effectively minimize the mortality and restore the altered hematological parameters and enhancing the innate immunity in O. fasciatus against E. tarda.     

Effects of Large-Scale Releases on the Genetic Structure of Red Sea Bream (Pagrus major,Temminck et Schlegel) Populations in Japan

Large-scale hatchery releases are carried out for many marine fish species worldwide; nevertheless, the long-term effects of this practice on the genetic structure of natural populations remains unclear. The lack of knowledge is especially evident when independent stock enhancement programs are conducted simultaneously on the same species at different geographical locations, as occurs with red sea bream (Pagrus major, Temminck et Schlegel) in Japan. In this study, we examined the putative effects of intensive offspring releases on the genetic structure of red sea bream populations along the Japanese archipelago by genotyping 848 fish at fifteen microsatellite loci. Our results suggests weak but consistent patterns of genetic divergence (F_ST = 0.002, p < 0.001). Red sea bream in Japan appeared spatially structured with several patches of distinct allelic composition, which corresponded to areas receiving an important influx of fish of hatchery origin, either released intentionally or from unintentional escapees from aquaculture operations. In addition to impacts upon local populations inhabiting semi-enclosed embayments, large-scale releases (either intentionally or from unintentional escapes) appeared also to have perturbed genetic structure in open areas. Hence, results of the present study suggest that independent large-scale marine stock enhancement programs conducted simultaneously on one species at different geographical locations may compromise native genetic structure and lead to patchy patterns in population genetic structure.     

Isolation and characterization of polymorphic microsatellite DNA markers in the rock bream (Oplegnathus fasciatus)

From a (GT)₁₃-enriched genomic library of Oplegnathus fasciatus, 14 polymorphic microsatellite were isolated and characterized in a test population with alleles ranging from two to nine, the observed and expected heterozygosities from 0.0000 to 1.0000, and from 0.1726 to 0.8507, respectively. Five loci deviated from the Hardy–Weinberg equilibrium, and linkage disequilibrium between two loci was significant. Two loci were also polymorphic in Pagrosomus major assessed for cross-species amplification. These polymorphic microsatellites will be useful for genetic diversity analysis and molecule-assisted breeding for O. fasciatus. Changwei Shao contributed equally.     

鳜鱼传染性脾肾坏死病毒(ISKNV)PCR检测方法的建立及虹彩病毒新证据

利用真鲷虹彩病毒（ＲＳＩＶ）核苷酸还原酶小亚单位（ＲＮＲＳ）基因保守区设计的一对引物,建立了鳜鱼传染性脾肾坏死病毒（ＩＳＫＮＶ）特异的ＰＣＲ检测体系。运用该体系检测ＩＳＫＮＶ,具有简便、快速、敏感、特异等特点,为诊断与预防ＩＳＫＮＶ提供了一个重要的手段。通过对ＰＣＲ产物的克隆与序列分析,发现ＩＳＫＮＶ ＰＣＲ扩增产物与ＲＳＩＶ ＲＮＲＳ基因相应序列的同源性很高,达到９２．５％,进一步证明ＩＳＫＮＶ     

Genomic Structure and Functional Analysis of Promoter Region of Somatolactin Gene of Sea Bream (<Emphasis Type="Italic">Sparus aurata</Emphasis>)

Somatolactin (SL) is a pituitary hormone belonging to the growth hormone–prolactin family and is produced in the intermediate lobe of teleosts. The SL gene was isolated from a sea bream genomic library and found to be composed of 5 exons distributed within a 9-kb length of DNA. Sequence analysis of the proximal promoter region showed the presence of a classical TATA box located 59 bp upstream from the initial start ATG codon, 5 consensus sequences corresponding to the Pit-1 binding element, and a putative CREB site. In CHO cells cotransfected with the DNA from 2 plasmids, one encoding sea bream Pit-1 under Rous sarcoma virus long terminal repeat regulation and one encoding the SL promoter driving the expression of luciferase, Pit-1 was found to enhance the expression of luciferase. Only one Pit-1 binding site was necessary for enhancement. Analysis by immunoblots of in vitro culture of pituitaries of Sparus aurata showed that several agents, including estradiol, verapamil, and phorbol myristate acetate, had different inhibitory effects on SL and growth hormone released to the culture medium.     

Ontogenetic change in the relationship between metabolic rate and body mass in a sea bream Pagrus major (Temminck & Schlegel)

Ontogenetic changes in the relationship between resting rate of oxygen consumption and wet body mass were examined at 20° C with the sea bream Pagrus major ranging from 0.00020 g (weight just after hatching) to 270 g (weight at 530 days old). There was a triphasic relationship between oxygen consumption of an individual fish M (μl min⁻¹) and body mass W (g). During a very early stage (weight 0.00020–0.00025 g), corresponding to the pre-larval stage and with the transitional period to the post-larval stage, there was no substantial change in body mass. The mass–specific metabolic rate M/W (μl g⁻¹ min ¹) showed no clear relationship to body mass as expressed by the equation M/ W =4.86 + 1.47 D , where D is age in days. During the post-larval stage (weight 0.00031–0.005 g), M/W remained almost constant independent of body mass following the expression M = 12.5 W0 .949. During the juvenile and later stages (weight 0.005–270 g), M/ W decreased with increasing body mass following the expression M = 6.3 W ^0.821 which is significantly different from the expression for the post-larval stage ( P < 0.001). Ontogenetic changes in the metabolism-body mass relationship are discussed from the viewpoint of relative growth of organs with different metabolic activities.     

Molecular cloning,characterisation and expression of the translationally controlled tumor protein gene in rock bream (Oplegnathus fasciatus)

Translationally controlled tumor protein (TCTP) is an important immune regulator that has been implicated in a number of cellular processes, including cell growth, cell cycle progression, apoptosis regulation and protection of cells against various environmental stresses. In this study, we cloned and characterised TCTP from rock bream (Oplegnathus fasciatus), which is an economically important species in the Korean aquaculture industry. The full-length rock bream TCTP (RbTCTP) cDNA was of 1,041 bp and contained an open reading frame of 513 bp, which encoded 170 amino acids. The 5′ untranslated region (UTR) was 90 bp, while the 3′ UTR was 438 bp, containing a polyadenylation signal. RbTCTP showed 76, 75 and 74 % amino acid sequence identities to those of tilapia (Oreochromis niloticus), orange-spotted grouper (Epinephelus coioides) and Japanese sea perch (Lateolabrax japonicus), respectively. The positions of microtubule binding region, Ca⁺ binding region and TCTP signature regions in RbTCTP were similar to other fish species and mammals. RbTCTP mRNA expression level was highest in the gill compared to other tissues. The level of RbTCTP mRNA expression was significantly regulated by injection of red seabream iridovirus, Streptococcus iniae and Edwardsiella tarda.